Bifunctional chelating agent for actinium

ABSTRACT

The invention concerns a ligand comprising (I) wherein n is an integer from 1 to 5 Y is CO2H or PO3H2T represents —X or -phenyl-X, wherein X represents NO2, NH2, NCS, NHCOCH2-Z, NHCO—W—COCNHS, —NH-Q, —NHCS-Q, —NHCOCH2-Q, or NHCO(CH2)m-Q where Q is a hapten chosen from the group consisting of steroids, enzymes, proteins, monoclonal antibodies, chimeric antibodies, or fragments thereof or any activated linker ready for coupling reaction, W is —(CH2)m- m is an integer from 1 to 10 Z is chloride, bromide or iodine.

TECHNICAL FIELD

[0001] The present invention relates to bifunctional chelating agentsand more particular to bifunctional polyaza polycarboxylic orpolyphosphonic macrocycles ligands a method of synthesis of theseproducts and their uses.

BACKGROUND OF THE INVENTION

[0002] As described by Davis, I. A. et al in the article “Comparison ofActinium 225 Chelates: Tissue Distribution and Radiotoxicity” publishedin Nucl. Med. Biol., Vol. 26, pp 581-589, 1999; by Deal, K. A. et al. inthe article “Improved in Vivo stability of Actinium-225 MacrocyclicComplexes” published in J. Med. Chem., Vol. 42, pp. 2988-2992, 1999, byGrote Gansey, M. H. B. et al. in the article “Conjugation,Immunoreactivity, and Immunogenicity of Calix (4) arenes; Model Study toPotential Calix (4) arenes-Based Ac3+ Chelators” published in Bioconj.Chem., Vol.10, pp 610-623, 1999, and by Ouadi, A. et al. in the article“Synthesis of a novel Bifunctional Chelating Agent for 225Accomplexation” published in Tet. Lett., Vol 41 pp 7202-7209, 2000, abifunctional chelating agent is necessary to bind with a good stabilitya radionuclide to a vector.

[0003] An object of the invention is to provide more effectivebifunctional chelating agents for metals especially actinides andlanthanides.

SUMMARY OF THE INVENTION

[0004] The present invention includes a ligand comprising:

[0005] wherein

[0006] n is an integer from 1 to 5

[0007] Y is —CO₂H or —PO₃H₂

[0008] T represents —X or -phenyl-X, wherein

[0009] X represents —NO₂, —NH₂, —NCS, —NHCOCH₂-Z, —NHCO—W—COCNHS, —NH-Q,—NHCS-Q, —NHCOCH₂-Q, or —NHCO(CH₂)_(m)-Q where Q is a hapten chosen fromthe group consisting of steroids, enzymes, proteins, monoclonalantibodies, chimeric antibodies, or fragments thereof or any activatedlinker ready for coupling reaction,

[0010] W is —(CH₂)_(m)—P1 m is an integer from 1 to 10

[0011] Z is chloride, bromide or iodine.

[0012] If X represents —NH-Q, —NHCS-Q, —NHCOCH₂-Q, or —NHCO(CH₂)_(m)-Qwhere Q is a hapten chosen from the group consisting of steroids,enzymes, proteins, monoclonal antibodies, chimeric antibodies, humanisedantibodies or fragments thereof, the resulting ligand is also called aligand-hapten conjugate.

[0013] The invention also includes according to a preferred embodiment,a metal chelate of the ligand as described above wherein the metal isactinium-225 (²²⁵Ac).

[0014] The present invention also includes the method of using the metalchelates of the ligand-hapten conjugate possessing a linking groupwherein the chelate as a therapeutic or diagnostic agent.

[0015] Specifically, such ligands are useful for radiolabeling proteinswith radioactive metals, and can consequently be utilised with respectto radioimmunoimaging and/or radioimmunotherapy. The presentligand-hapten conjugates firmly link actinium to proteins, minimisemetal release and permit high selective delivery of metals to targetedsites in vivo. This is especially true for the actinium complexationmetal chelate protein conjugates.

[0016] Immunotherapy with radiolabelled antibodies allows fairlyspecific targeting of certain cancers (see e.g. Couturier, O. et al.“Validation of 213-Bi-alpha radioimmunotherapy for multiple myeloma” inClin. Cancer. Res., Vol. 5, pp. 3165-3170, 1999; Huneke, R. B., et al.in “Effective alpha-particle-mediated radioimmunotherapy of murineleukemia” in Cancer Res., Vol. 52, pp. 5818-5820, 1992; Kennel, S. J. etal. “Radioimmunotherapy of micrometastases in lung with vasculartargeted 213Bi” in Br. J. Cancer, Vol. 80, pp. 175-184, 1999; Kozak, R.W. et al. “Bismuth-212-labeled anti-Tac monoclonal antibody:alpha-particle-emitting radionuclides as modalities forradioimmunotherapy” in Proc. Natl. Acad. Sci. USA, Vol. 83, pp. 474-478,1986 or Macklis, R. M. et al. “Radioimmunotherapy withalpha-particle-emitting immunoconjugates” in Science, Vol. 240, pp.1024-1026, 1988).

[0017] This technique is based on the use of radionuclides associated toantibodies or peptides that are specific of antigens expressed on thetumor cells. In order to bind a radionuclide to a vector it is necessaryto use bifunctional chelating agents (BCA) that have two specific sites.One site is to be coupled to the vector and the other has to form verystable complexes with the radionuclide to be used.

[0018]²²⁵Ac, which is an alpha emitter, is a good candidate for suchapplications as described by Kaspersen, F. M. et al. “Cytotoxicity of213Bi- and 225Ac-immunoconjugates” in Nucl. Med. Commun., Vol. 16, pp.468-476, 1995. The very short range (<100 μm) of α-particles and thehigh energy transfer allows efficient destruction of tumor cells whereasnormal cells are relatively spared.

[0019] Chelators that can hold radioactive metals with high stabilityunder physiological conditions are essential to avoid excessiveradiation damage to non-target cells.

[0020] Furthermore, these bifunctional chelating agents allow differentapplications; it can be used to bind ²²⁵Ac to any biological ornon-biological structures for any applications.

[0021] These chelating agents can be used non-associated to a vector asa detoxication chelating agent or using the natural tropism of thecomplex.

[0022] This chelating agent can also be used grafted on achromatographic column in order to purify or concentrate any solutionscontaining ²²⁵Ac.

[0023] The complexation properties of our product with ²²⁵Ac show thatthis chelating agent may also be useful as a good extractant in theprocess of separation of minor actinides and lanthanides in nuclearwaste or to separate specific groups of metals in high level waste.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Embodiments of the present invention are described by way ofexample and with reference to the accompanying drawing wherein:

[0025] An access route that allows the synthesis of a bifunctionalmacrocycle chelating agent is described in FIG. 1.

[0026]FIG. 1 represents a scheme for the preparation of a substituted1,4,7,10,13,16,19,22-octa(2-carboxymethyl)-octaaazacyclotétracosaneligand (8).

DETAILED DESCRIPTION

[0027] Different non-functionalised chelating agents (commerciallyavailable or readily synthesised in the laboratory) bearingaminocarboxylate groups (EDTA, DTPA, DOTA, PEPA, HEHA and HOHEC) oraminophosphonate groups (EDTMP) were tested for their complexationproperties with ²²⁵Ac. It was found that HOTEC compound (1,4,7,10,13,16,19,22-octacarboxylmethyl-1,4,7,10,13,16,19,22-octaazacyclotetracosane)appeared to be the best candidate for ²²⁵Ac complexation. This result isin balance in regard of previous studies. Polyaza polycarboxylicmacrocycles are known to form thermodynamically stable complexes withlarge metal ions such as actinides and lanthanides.

EXAMPLE

[0028] N-((Methoxycarbonyl)methyl)-4-nitrophenylalanine methyl ester (2)

[0029] Triethylamine (22 mmol) was added to a suspension of4-nitrophenylalanine methyl ester hydrochloride (1) (21 mmol) in THF (50ml). The mixture was stirred at room temperature for one hour, thetriethylamine hydrochloride was filtered off, and the filtrateconcentrated to yellow oil. The oil was dissolved in dry THF (50 ml) andto this solution was added triethylamine (60 mmol) andmethylbromoacetate (60 mmol), the solution was stirred at roomtemperature under nitrogen atmosphere for 3 hours, after which theprecipitate was filtered off and the filtrate concentrated on vacuum.The residue was dissolved in ethylacetate, washed with H₂O, dried(MgSO₄) and concentrated on vacuum to give yellow oil.

[0030] Compound (3)

[0031] Sodium (20 mmol) was dissolved in dry methanol (100 ml) at roomtemperature under nitrogen atmosphere and to this solution was addedhexaethyleneheptamine (18 mmol) andN-((methoxycarbonyl)methyl)-4-nitrophenylalanine methyl ester (2) (18mmol). This solution was refluxed for 72 hours after which the solventwas removed and the residue was purified on silica gel chromatographywith chloroforme/methanol/NH₃ (aq) (75:20:5).

[0032] Compound (4)

[0033] A solution of BH₃ in THF (100 mmol) was added dropwise to astirred suspension of (3) (10 mmol) in THF (50 ml) at 0° C. undernitrogen atmosphere. The solution was heated at reflux for 36 hours.Methanol was added slowly to the solution at 0° C. after which thesolvent was removed and the residue was dissolved in methanol (50 ml);the resulting mixture was cooled at 0° C. and gaseous HCl was bubbledthrough the solution and then the mixture was refluxed for 12 hours. Theresulting precipitate was collected washed with ether to give a whitepowder. The solid was dissolved in water and was loaded on a column ofDOWEX 1X-8 anion-exchange resin (OH⁻form). The column was eluted withwater; alkaline fractions were collected, and the water was removedunder vacuum

[0034] Compound (5)

[0035] To a solution of (4) (10 mmol) in DMF (50 ml) at room temperatureunder a nitrogen atmosphere was added anhydrous sodium carbonate (0.11mol) and a solution of tert-butyl bromoacetate (62 mmol) in DMF (20 ml).The mixture was heated at 60° C. for 18 hours after which the solventwas removed and the residue was dissolved in chloroform washed withbrine, dried (MgSO₄) and concentrated on vacuum.

[0036] Compound (6)

[0037] To a solution of (5) (15 mmol) in ethanol (50 ml) at roomtemperature was added Pd/C under H₂ atmosphere. The mixture was refluxedfor 2 hours, the residue was passed over Celite. The filtrate wasevaporated on vacuum.

[0038] Compound (8)

[0039] (10 mmol) of (6) was treated with TFA (0.1 mol) 6 hours at roomtemperature under nitrogen atmosphere after which the solvent wasremoved.

[0040] The compound thus obtained was dissolved in water and loaded on acolumn of DOWEX 50WX8-200 (H⁺form).

[0041] The column was eluted consecutively with 0.5 M HCl and with wateruntil the eluent was neutral and finally with 0.5 M aqueous ammoniasolution. Alkaline fractions were collected, and the water was removedon vacuum

[0042] The compound thus obtained was dissolved in water and the pH wasadjusted to 9.0 with NaHCO₃. To this solution was added at roomtemperature under nitrogen atmosphere thiophosgene in CHCl₃ (10 ml), themixture was stirred for 2 hours. The organic layer was removed and thewater was evaporated on vacuum to give the final product (8).

[0043] While a preferred embodiment of the present invention has beendescribed, it will apparent to those skilled in the art that manychanges and modifications may be made without departing from theinvention in its broader aspects. The appended claims are thereforeintended to cover all such changes and modifications as fall within thetrue spirit and scope of the invention.

1. A ligand comprising

wherein n is an integer from 1 to 5 Y is —CO₂H or —PO₃H₂ T represents —Xor -phenyl-X, wherein X represents —NO₂, —NH₂, —NCS, —NHCOCH₂-Z,—NHCO—W—COCNHS, —NH-Q, —NHCS-Q, —NHCOCH₂-Q, or —NHCO(CH₂)_(m)-Q where Qis a hapten chosen from the group consisting of steroids, enzymes,proteins, monoclonal antibodies, chimeric antibodies, or fragmentsthereof or any activated linker ready for coupling reaction, W is—(CH₂)_(m)— m is an integer from 1 to 10 Z is chloride, bromide oriodine.
 2. A metal chelate of the ligand of claim 1, wherein the metalis actinium.
 3. A metal chelate of the ligand of claim 2, wherein themetal is actinium-225 (²²⁵Ac).
 4. A metal chelate of the conjugate ofclaim 2, for use as a therapeutic or diagnostic agent. 5-8. (Cancelled).9. A medicament, comprising: the metal chelate as claimed in claim 2.10. A nuclear waste extraction agent, which comprises: the ligand ofclaim 1; wherein said ligand optionally comprises actinium oractinium-225.
 11. A solid chromatographic support, comprising: theligand of claim 1; wherein said ligand optionally comprises actinium oractinium-225.
 12. A detoxication agent, which comprises: the ligand ofclaim 1; wherein said ligand optionally comprises actinium oractinium-225.